1. Field of the Invention
The present invention relates to a specimen holder, specimen inspection apparatus, and specimen inspection method capable of observing or inspecting a specimen consisting of cultured tissues and cells of an animal or plant.
2. Description of Related Art
In the fields of life science and pharmaceutics, it has become important to observe reactions of biological cells produced by giving a stimulus (such as electricity, chemical substance, or medicine) to them. In the past, optical microscopes have been used for such observation. Manipulators or pipettes have been employed to give stimuli to cells. Frequently, important portions to be observed are very tiny regions of less than 0.1 μm that are impossible to observe with an optical microscope.
For example, diseases arising from the inability to exchange substances normally among biological cells include hypertension, diabetes insipidus, arrhythmia, myopathy, diabetes, and deprementia. Exchange of substances among cells is performed by ion channels having sizes of about 10 nm and existing in cell membranes. Because it is difficult to observe such ion channels with optical microscopes, there has been a demand for a technique enabling observation using a scanning electron microscope (SEM) having high resolution.
However, a specimen to be inspected with an inspection apparatus incorporating SEM capabilities is normally placed in a specimen chamber whose internal pressure has been reduced by vacuum pumping. The specimen placed in the specimen chamber, which, in turn, is placed in a reduced-pressure ambient in this way, is irradiated with an electron beam (charged-particle beam). Secondary signals, such as secondary electrons or backscattered electrons, produced from the specimen in response to the irradiation are detected.
In such inspection of a specimen using SEM, the specimen is exposed to a reduced-pressure ambient. Therefore, moisture evaporates from the specimen, so that the cells die whereupon it has been impossible to observe reactions of living cells to a stimulus.
When a specimen consisting of dead cells where the proteins have been fixed is placed in a reduced-pressure ambient or vacuum ambient, much labor and various pretreatments, such as dehydration, drying, and metal vapor deposition, that require a high degree of skillfulness have been necessary to prevent evaporation of moisture within a vacuum; otherwise, the specimen would be deformed. Accordingly, an excessively long time has been required to observe the specimen. It has not been possible to achieve high throughput observations.
For these reasons, in order to observe a biological specimen, it is desired to prevent evaporation of moisture. When an inspection is performed under the condition where the specimen contains moisture, it is necessary to prevent the specimen from being exposed to the reduced-pressure ambient; otherwise, moisture would evaporate from the specimen. One conceivable method of inspecting a specimen using SEM without exposing the specimen to a reduced-pressure ambient in this way consists of preparing a specimen holder (sample capsule) whose opening (aperture) has been sealed off by a film, placing the specimen in the holder, and installing the holder in an SEM specimen chamber that is placed in the reduced-pressure ambient.
The inside of the specimen holder in which the specimen is placed is not evacuated. The film that covers the opening formed in the sample capsule withstands the pressure difference between the reduced-pressure ambient inside the SEM specimen chamber and the ambient (e.g., atmospheric-pressure ambient) of the inside of the specimen holder that is not pumped down. Furthermore, the film permits an electron beam to pass therethrough (see JP-T-2004-515049).
When a specimen is inspected, an electron beam is directed at the specimen placed within the sample capsule from outside the capsule via the film on the capsule. The capsule is placed in the SEM specimen chamber at a reduced-pressure ambient. Backscattered electrons are produced from the irradiated specimen. The backscattered electrons pass through the film on the capsule and are detected by a backscattered electron detector mounted in the SEM specimen chamber. Consequently, an SEM image is derived.
However, with this technique, the specimen is sealed in the closed space and thus it has been impossible to give a stimulus to cells from the outside with a manipulator or the like. Where the cells should be observed or inspected in vivo for a long time after the specimen consisting of the cells has been sealed in the sample capsule, there arises a problem.
Furthermore, an SEM image has high resolution but contains only black-and-white information. Therefore, it has been difficult to identify the observed tissue. On the other hand, in optical microscopy, fluorescence labeling technology has been established, and it is easy to identify tissues. If an SEM image and an optical microscope image derived from a substantially identical position can be observed substantially at the same time, the tissues can be identified with the high-resolution SEM image. However, with the aforementioned sample capsule, it is necessary to open the capsule for obtaining an optical microscope image. In order to derive an SEM image, it is necessary to close the capsule. Hence, it has been impossible to achieve the simultaneous observation.
Usually, cells are cultured by adsorbing them onto a laboratory dish having a diameter of more than 35 mm, pouring a culture medium onto the dish, and culturing the cells under conditions including a temperature of 36° to 38° C. (normally, 37° C.) and a carbon dioxide concentration of 3% to 10% (normally, 5%). When the cells are observed, the cells are peeled off from the dish and put into the sample capsule.
However, the environment inside the sample capsule is different from the environment on the laboratory dish and so the possibility that cells survive within the specimen container is low. That is, with the sample capsule described in JP-T-2004-515049, only about 15 μl of solution can be put into it. Because the environmental ambients including pH and osmotic pressure vary in a short time, it has been difficult to culture cells.
Examples of a method of acquiring an SEM image by irradiating a specimen with an electron beam via a film capable of withstanding the pressure difference between vacuum and atmospheric pressure in this way and detecting backscattered electrons emanating from the specimen are also described in “Atmospheric scanning electron microscopy,” Green, Evan Drake Harriman, Ph.D., Stanford University, 1993 (especially, Chapter 1: Introduction) and JP-A-51-42461.
Examples in which two films of the structure described above are placed opposite to each other with a specimen interposed between the films and in which an image is acquired by a transmission electron microscope are described in JP-A-47-24961 and JP-A-6-318445. Especially, JP-A-47-24961 also states a case in which an SEM image of the specimen interposed between such films is acquired.
A morphological variation caused by a reaction occurring in a cell after a stimulus is given to the cell using a manipulator or pipette takes place in a very tiny region within the cell and, therefore, the variation cannot be observed with an optical microscope. High resolution imaging using SEM is essential. In order to observe cells by SEM while maintaining the liquid, the specimen (cells) cultured on a laboratory dish is sealed into a sample capsule, and then the specimen is irradiated with an electron beam via a film formed on the capsule so as to image the specimen.
However, the sample capsule is a closed space. This makes it impossible to use a manipulator or pipette for giving a stimulus. Furthermore, the probability that cells sealed in sample capsules survive has been low. In addition, even if high resolution imaging is enabled by SEM, it is impossible to identify tissues. It is desired to observe the tissues with an optical microscope simultaneously because the optical microscope permits identification of the tissues.
Consequently, there is a demand for development of a specimen holder which permits cultured cells to be manipulated from the outside or a chemical or medicinal solution to be spread over the cells and which enables a user to well observe and inspect the cells in vivo.
In the above-described specimen holder, it is desired that cells can be cultured for a long time. This enables the specimen to be well observed or inspected in vivo. Additionally, simultaneous observation with an optical microscope and an SEM is preferably enabled such that a stimulus can be given to cultured cells using a manipulator or pipette and that the specimen can be observed or inspected well.
Especially, in a case where a chemical is sprayed over cultured cells using a manipulator or pipette, it is desired to reduce the amount of the sprayed chemical.